Abstract
The Locus Control Region (LCR) requires intronic elements within β-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional β-globin intron 2 elements that rescue LCR activity directed by 5′HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igμ 3′MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igμ 3′MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5′HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5′HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.
Highlights
The b-globin gene is regulated by a Locus Control Region (LCR) that interacts with gene proximal elements to activate erythroid specific expression
Many studies demonstrate that individual LCR hypersensitive sites (HS) loop out the intervening DNA to interact with the globin genes [1,2,3,4], and that transcriptional activation is accompanied by movement of the gene away from heterochromatin at the nuclear periphery towards transcription factories located more centrally [5,6,7,8]
The immunoglobulin m (Igm) 39MAR is able to transmit through a lentivirus and provides position independent expression in primary B lymphocytes [41]. These findings suggest that the Igm 39MAR might perform the same function when substituted for the b-globin ATR, and that this function could be transmitted through a lentivirus vector
Summary
The b-globin gene is regulated by a Locus Control Region (LCR) that interacts with gene proximal elements to activate erythroid specific expression. While the LCR is not required to establish open chromatin at the endogenous locus [9], it is able to open chromatin at ectopic bglobin transgene integration sites to activate high level transcription [10,11,12]. This ability of the LCR, in particular its 59HS3 element [13], to open chromatin at all integration sites has made it a widely used component of gene therapy expression cassettes designed for treatment of b-thalassemia or Sickle Cell Anemia. In order to reduce potential genotoxicity, it would be advantageous to include insulator elements in the LTRs to Author Summary
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